This is a competing renewal of 5R01 HL034708-22, which has supported the development of a clinically relevant model of cardiac preconditioning using human cardiomyocytes derived from the human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), derived from both normal iPSCs (N-iPSCs) and type 2 diabetes mellitus iPSCs (DM-iPSCs). In the past funding cycle, focus has been on using human cardiac tissue obtained during cardiac bypass; in those studies we investigated the underlying mechanisms of volatile anesthetic-induced cardiac protection from ischemia/reperfusion (I/R) injury. However, our approach had several limitations, including the use of atrial specimens from patients exhibiting various diseases and in whom different drug therapies were utilized. This in vitro model of human disease will enable developmental and comparative studies of normal and diabetic cardiomyocytes to address genetic and environmental mechanisms responsible for attenuation of preconditioning efficacy in diabetics. Importantly, the proposed experiments will also yield new insights into how diabetes might alter the potential efficacy of stem cells for future use in regenerative medicine. The working hypothesis is that delayed opening of the mitochondrial permeability transition (PT) pore during I/R is central for APC, and that diabetes impairs cardioprotection through actions on mitochondria that are both acute (hyperglycemia) and genetic in origin. On the basis of our progress in developing a clinically relevant model of cardiac preconditioning, we propose the following Specific Aims: Specific Aim 1. Determine mitochondrial bioenergetics, ion homeostasis, and signaling pathways in human ventricular cardiomyocytes derived from N-iPSCs and DM-iPSCs. Specific Aim 2. Determine contributions of sarcKATP channel to anesthetic-induced mitochondrial protection of human cardiomyocytes derived from N-iPSCs and DM-iPSCs. Specific Aim 3. Determine how anesthetics modulate human PT pore opening under I/R stress in intact human ventricular cardiomyocytes derived from N-iPSCs and DM-iPSCs. In summary, the goal of this proposal is to utilize the in vitro differentiation of human embryonic and induced pluripotent stem cells into cardiac lineage to delineate the genetic vs. environmental mechanisms responsible for the lack of efficacy of APC in diabetes. Our preliminary data indicate that the cardiomyocytes derived from iPSCs from normal and diabetic patients exhibit functional, structural, and molecular properties of early-stage human myocytes. These studies will provide novel mechanistic information on the roles of diabetes and hyperglycemia in modulating anesthetic-induced cardioprotection through changes in mitochondrial function, protein phosphorylation, altered ROS formation, and KATP channel activity. Completion of the specific aims will elucidate the novel role of mitochondria to modulate APC during diabetes and in the long run, may suggest new therapeutic targets for perioperative intervention.